Sequence context dependent (SCD) effects are involved in the process of mutagenesis and define mutational hotspots in tumors, but little is known about such effects, except as mutational outcomes. The experiments in this proposal will rigorously examine SCD effects in excision and mutagenesis that would contribute to mutational hotspots. Initially, we will establish SCD rules for DNA glycosylase excision. Once the rules are elucidated, our hypotheses regarding the physical-chemical factors governing SCD excision rules for DNA glycosylases will mold the experimental design. Finally, by varying the sequence contiguous to a unique lesion, we will evaluate the role of SCD effects on mutagenesis. The studies proposed will answer important biological questions concerning factors influencing mutation, the quality of mutation databases for tumors, and the mutability of a particular base that could lead to cancer.